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R F Murphy

Publications and source records attributed to R F Murphy.

At least 109 records · Page 6Linked to original sources
PubMed1990

Endosomes can undergo an ATP-dependent density increase in the absence of dense lysosomes.

On the basis of evidence that lysosomal enzymes and membrane proteins are present in endosomes, we have previously suggested that the production of lysosomes involves maturation rather than vesicle fusion (Roederer, M., R. Bowser, R. F. Murphy, J. Cell. Physiol. 131, 200-209 (1987)). Since the appearance of endocytosed material in lysosomes is associated with an increase in buoyant density from that of endosomes, a prediction of the model is that endosomes should be capable of undergoing such an increase in vitro. We observe that under appropriate conditions, isolated endosomes containing [125I]EGF can undergo an increase in density in vitro to that of dense lysosomes, mimicking the density change which occurs in vivo. This occurs in the absence of dense lysosomes with which to fuse. The density increase requires ATP and can be efficiently inhibited in vitro by the presence of benzylamine, suggesting that vesicular acidification is required. Since low pH has previously been shown to induce formation of a matrix by lysosomal enzymes in vitro (Buckmaster, M. J., A. L. Ferris, B. Storrie, Biochem. J. 249, 921-923 (1988)), we propose that a mechanism by which endosomes and/or lysosomes increase their density is a low pH induced aggregation of vesicle contents which decreases the osmotic pressure inside the vesicle. Together with previous data, the results provide highly suggestive evidence that the pathway to lysosomes includes a maturation of the postsorting compartment into what has classically been termed a lysosome.

Adenosine Triphosphate↗
PubMed1989

Flow cytometry DNA ploidy analysis and catecholamine secretion profiles in neuroblastoma.

Previous studies have shown that catecholamine secretion patterns have been imperfect predictors of clinical behavior of neuroblastomas. Recently, studies of nuclear DNA content in neuroblastoma have shown that an aneuploid DNA content predicts favorable clinical behavior. To determine if a correlation exists between these tumor biologic indicators, the authors analyzed both in a series of 39 patients with neuroblastoma. Flow cytometric DNA analysis performed on paraffin blocks determined that 23 patients had tumors with aneuploid DNA content (aneuploid tumors) and 16 patients showed no demonstrable anomalies of tumor DNA content (nonaneuploid tumors). Comparison of catecholamine levels in urine and tumor homogenates with DNA content data indicate that nonaneuploid neuroblastomas include a significant number (P less than 0.02) of biochemically primitive tumors which secrete high levels of 3,4 dihydroxyphenylalanine (DOPA), dopamine and homovanillic acid (HVA). This suggests a dopamine-norepinephrine pathway block, which supports previous reports of deficiency of dopamine beta-hydroxylase activity in some neuroblastomas. The study shows that in contrast to aneuploid tumors, nonaneuploid neuroblastomas secrete higher levels of early pathway catecholamine metabolites and are more likely to present in higher (unfavorable) clinical stages of disease.

Aneuploidy↗
PubMed1989

The pharmacokinetics of zidovudine administered by continuous infusion in children.

STUDY OBJECTIVE: To define the pharmacokinetics of zidovudine (azidothymidine) in children with human immunodeficiency virus infection. DESIGN: Plasma, urine, and cerebrospinal fluid were obtained following a single 80 mg/m2 body surface dose infused over 1 hour (n = 9), and during a continuous infusion of 0.5 (n = 3), 0.9 (n = 8), 1.4 (n = 7), or 1.8 (n = 3) mg/kg body weight per hour. SETTING: Outpatient clinic and inpatient ward of the Pediatric Branch of the National Cancer Institute. PATIENTS: Twenty-one children (seventeen boys) ranging in age from 14 months to 12 years with symptomatic human immunodeficiency virus infection who were being treated on a phase I-II study of continuous intravenous infusion zidovudine. MEASUREMENTS AND MAIN RESULTS: Zidovudine disappearance following bolus administration was rapid and biexponential with half-lives of 9.6 and 92 minutes, and a total clearance of 705 +/- 330 mL/min.m2. Zidovudine remained above 1 mumol/L, the optimal virostatic concentration in vitro, for only 1.5 hours. In contrast, with continuous infusion steady-state plasma zidovudine concentrations (Css) were maintained above 1 mumol/L continuously, even at the lowest infusion rate. At steady state the ratio of cerebrospinal fluid zidovudine concentration to plasma was 24% +/- 9%. Patients who developed severe neutropenia (absolute neutrophil count less than 0.5 X 10(9)/L) on the continuous infusion regimen had significantly higher plasma Css. Six of eight had a Css greater than 3.0 mumol/L. CONCLUSIONS: Pharmacokinetic parameters show that continuous infusion is better than an intermittent schedule in maintaining minimal virostatic concentrations of the drug with a lower daily dose.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Action of amino acids on stomach, pancreas, and gallbladder in dogs.

An intravenous infusion of a 3% solution of amino acids was given to 8 dogs, all with Heidenhain pouches and gastric fistulae. Four of these had duodenal cannulae opposite the pancreatic and 4 opposite the biliary ampulla. The usual basal 100 min spontaneous peaking of pancreatic juice volume and protein secretion was seen but peaks were abolished by the amino acid infusion and troughs were slightly elevated, but the total 90 min volume protein and bicarbonate outputs were not different from control. Gastric acid and pepsin secretions were augmented reaching a peak during the first hour with a subsequent decline. The 90 min acid and pepsin output was significantly higher than control. The gallbladder contracted during the first hour and remained thus until the infusion was terminated. This happened even when the duodenum was kept alkaline, but was abolished by ganglionic blockade. During intravenous amino acid infusion the patterns of gallbladder activity and pancreatic secretion resembled those of the post cibal rather than fasting state.

Amino Acids↗
PubMed1989

Growth-inhibitory and growth-stimulatory effects of epidermal growth factor on human breast cancer cell line, MDA.MB.436: dependence on culture conditions.

Epidermal growth factor (EGF) is growth inhibitory for some cell lines, especially those having over-expressed EGF receptors. We have examined the effects of murine EGF on the growth of the human breast cancer cell line, MDA.MB.436, which has low numbers of EGF receptors. In the presence or absence of serum a 6 day exposure to 0.1 ng/ml EGF causes inhibition of growth if the culture medium is left unchanged during the course of the experiment but the same concentration of EGF causes stimulation above control if the EGF-containing medium is replaced daily. A 1 day exposure to 0.1 ng/ml followed by return to control medium has no effect on subsequent growth. The cells do not synthesize EGF receptor binding activity and added EGF is degraded within 2 days, suggesting that the inhibitory effects of EGF persist in its absence.

Breast Neoplasms↗
PubMed1989

Pharmacokinetics of zidovudine administered intravenously and orally in children with human immunodeficiency virus infection.

Zidovudine pharmacokinetics were determined in 16 children with human immunodeficiency virus infection who were being treated intravenously and orally on an intermittent schedule (every 6 hours). The intravenous doses studied were 80 (n = 3), 120 (n = 4), and 160 (n = 5) mg/m2/dose, infused over 1 hour. Fourteen patients were monitored after an oral dose of zidovudine at 120 (n = 2), 180 (n = 7), or 240 (n = 5) mg/m2/dose. Zidovudine was assayed with a reverse-phase high-pressure liquid chromatography method. Zidovudine disappearance after intravenous administration was rapid and biexponential, with half-lives of 14 and 90 minutes and a total clearance of 641 +/- 161 ml/min/m2. The volume of distribution at steady state was 45 +/- 28 L/m2. These pharmacokinetics parameters are very similar to those reported in adults. When administered orally, zidovudine was rapidly absorbed. The fraction of the oral dose that was bioavailable was 0.68 +/- 0.25, so that a 50% increment in the dose, in the conversion from intravenous to oral administration, resulted in plasma zidovudine concentrations after oral dosing that were nearly identical to those achieved with the 1-hour intravenous infusion. However, a dose of 180 mg/m2 given orally every 6 hours maintained plasma zidovudine concentrations in the target range of 1 mumol/L for less than half of the dosing interval. Other schedules, routes of administration, or oral drug formulations may have to be considered if sustained continuous exposure to micromolar zidovudine concentrations is desired.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Synthesis of somatostatin by breast cancer cells and their inhibition by exogenous somatostatin and sandostatin.

Three human breast cancer cell lines ZR-75-1, MDA-MB-436 and MCF-7 were found to contain respectively, 3.06, 2.69 and 1.86 fmol of somatostatin-like immunoreactivity (SLI) per 10(6) cells. Since SLI is undetectable in the passaging media it must, therefore, be synthesised by the cells. In the presence of fetal calf serum the cells were growth inhibited by addition of somatostatin or its long-lasting analogue, Sandostatin, but only after 3 days of continuous exposure. A 1-day exposure to either peptide had little or no effect on subsequent cell growth in peptide-free medium. Inhibition of cell proliferation is not due to cytotoxic effects of the dose used (500 ng ml-1, each) since both peptides caused short-term stimulation of growth in the absence of serum.

Breast Neoplasms↗
PubMed1989

Assay of vacuolar pH in yeast and identification of acidification-defective mutants.

As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification. Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo. Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vph1-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pep12::LEU2 mutant appeared defective by fluorescence-ratio microscopy and quinacrine-uptake assays, but the vacuolar pH in the pep12::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.

Hydrogen-Ion Concentration↗
PubMed1989

Regulation of endocytic pH by the Na+,K+-ATPase in living cells.

Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.

Animals↗
PubMed1989

Bioactivity studies on new gastrin analogues.

Gastrin antagonists may be useful in the treatment of peptic ulcer disease and gastrointestinal malignancies. The aim of the present study was to synthesize gastrin analogues and test them for their ability to inhibit gastrin-stimulated acid secretion. Five peptides were synthesized: peptide [2], in which methionine was replaced by leucine, and the COOH-terminal amide was replaced by the thiomethylamide; peptide [3], in which the COOH-terminal phenylalanine was removed, and the aspartic acid thioamidated; peptide [5], in which methionine was replaced by leucine, and the peptide bond between leucine and aspartic acid was replaced by a thioamide; peptide [7], in which the bond between leucine and aspartic acid was replaced by a ketomethylene amino bond; and, finally, peptide [8], in which a beta-bend was induced in the COOH-terminal region by the introduction of a D-phenylalanine in place of glycine. The biologic effect of the peptides was tested in the totally isolated, vascularly perfused rat stomach. The peptides were tested in concentrations of 10(-9), 10(-7), and 10(-5) M for agonist activity and together with gastrin 1-17, 5.2 X 10(-10) M, at a concentration of 10(-5) M for antagonist activity. Peptide [2] had full biologic activity but greatly reduced potency, and peptide [7] had a faint biologic activity. None of the peptides showed any antagonist activity.

Amino Acid Sequence↗
PubMed1989

Effect of confluence on endocytosis by 3T3 fibroblasts: increased rate of pinocytosis and accumulation of residual bodies.

We have compared lysosomal enzyme distributions on density gradients and rates of transport of endocytic markers for actively-growing and confluent cells. While it has been previously established that mammalian cells accumulate lysosomal enzymes during quiescence, we show that this accumulation is predominantly in residual bodies (p greater than 1.12 g/ml) rather than in dense lysosomes (p = 1.08-1.10 g/ml) and does not represent a change in the endosomal and lysosomal enzyme content. The accumulation is not caused by a change in the rate of production of dense lysosomes, since the rate of transfer of epidermal growth factor (EGF) from light to dense compartments is the same between confluent and subconfluent cells. Confluent cultures have a higher rate of initial pinocytosis, and a higher rate of retroendocytosis and/or recycling, causing a net lower rate of accumulation of fluid-phase material. The accumulation of residual bodies in confluent cultures may be caused by a lower rate of exocytosis of their contents and/or a lack of dilution by cell division. The data indicate that the impact of culture confluence must be carefully assessed in experiments designed to analyze endocytic pathways.

Animals↗
PubMed1989

Chronopharmacokinetics of oral methotrexate and 6-mercaptopurine: is there diurnal variation in the disposition of antileukemic therapy?

The chronopharmacokinetics of the orally administered antileukemic drugs, 6-mercaptopurine and methotrexate, were examined in 13 children with acute lymphoblastic leukemia (ALL) to establish if there is a pharmacokinetic basis for the lower relapse rate associated with administration of these agents in the evening. Children with ALL in complete remission had plasma drug concentrations monitored for 8 h following an oral dose of either methotrexate or 6-mercaptopurine administered in the morning (8 a.m.) and the evening (8 p.m.). Total drug exposure to oral methotrexate, as measured by the mean area under the plasma concentration-time curve (AUC), was 2.75 microM.h following the morning dose and 2.77 microM.h in the evening. For 6-mercaptopurine, the mean morning AUC (198 ng.h/ml) was higher than that following the evening dose (167 ng.h/ml) (p greater than 0.05); but compared to the wide interpatient variability observed with this drug, this 20% difference is not likely to be clinically significant. These results indicate that the suggested benefit of evening drug administration is not likely to be a result of diurnal variation in drug disposition.

Administration, Oral↗
PubMed1988

Effect of continuous intravenous infusion of zidovudine (AZT) in children with symptomatic HIV infection.

To produce concentrations of zidovudine (AZT) in plasma and cerebrospinal fluid that would provide constant inhibition of the replication of human immunodeficiency virus (HIV), we gave AZT by continuous intravenous infusion to 21 children ranging in age from 14 months to 12 years who had acquired HIV infection through transfusions or perinatally. All patients were symptomatic before AZT treatment (Class P2 of the Centers for Disease Control); 13 (62 percent) had evidence of neurodevelopmental abnormalities. The mean CD4/CD8 ratio was 0.18; 11 patients had CD4 counts below 0.2 x 10(9) per liter. We administered AZT at four dose levels: 0.5, 0.9, 1.4, and 1.8 mg per kilogram of body weight per hour. The plasma drug concentrations achieved at the respective dose levels were 1.9 +/- 0.3, 2.8 +/- 1.4, 3.1 +/- 1.1, and 4.5 +/- 1.0 microM. The steady-state cerebrospinal fluid:plasma ratio was 0.24 +/- 0.07. The only evidence of toxicity was bone marrow suppression. Transfusion was required in 14 patients because of low levels of hemoglobin (5 mmol per liter [less than 8 g per deciliter]). Dose-limiting neutropenia (less than 0.5 x 10(9) polymorphonuclear leukocytes per cubic millimeter) occurred in most patients who received doses of 1.4 mg per kilogram per hour or more. Improvement in neurodevelopmental abnormalities occurred in all 13 children who had presented with encephalopathy before treatment. Serial measurements of IQ before therapy and after three and six months of continuous therapy with AZT showed that IQ scores, including those for verbal and performance IQ, rose in these 13 patients and in 5 other children who had no detectable evidence of encephalopathy before treatment. Most patients also had increased appetite and weight, decreased lymphadenopathy and hepatosplenomegaly, decreased immunoglobulin levels, and increased numbers of CD4 cells. In some patients the improvement in the features of encephalopathy occurred despite the absence of immunologic improvement. We conclude that AZT is beneficial in children with symptomatic HIV infection, especially those with encephalopathy (which may be subclinical), and that the optimal continuous intravenous dose of AZT in children is between 0.9 and 1.4 mg per kilogram per hour.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Flow cytometric DNA analysis of neuroblastoma and ganglioneuroma. A 10-year retrospective study.

Retrospective quantitative DNA analysis was done on 147 samples from 89 patients with neuroblastoma and ganglioneuroma using flow cytometry. In the neuroblastoma patients, nuclear DNA content was found to be a stable tumor marker irrespective of site (primary versus metastatic) and despite changes with time in tumor progression, maturation, or therapy. The occurrence of DNA aneuploidy, which was detected in 60% of the neuroblastoma patients, paralleled other favorable indicators and was highly associated with survival (P less than 0.001). Of clinical stage, age, primary site, sex, and DNA content, only stage and DNA content correlated with survival. Those patients with favorable stage and DNA aneuploidy had higher survival rates. Further, favorable stage and the presence of DNA aneuploidy were independent prognostic indicators. Abnormal DNA content was also detected in samples from ganglioneuromas in which significant numbers of ganglion cell nuclei were recovered. These results indicate a striking difference between neuroblastoma and adult tumors in which DNA aneuploidy is generally a poor prognostic sign and provide a molecular link between ganglioneuromas and their malignant counterparts.

Adolescent↗
PubMed1988

Gastrin-releasing peptide (GRP) immunoreactivity in the rat retina: a radioimmunoassay, immunohistochemical and chromatographic study.

Using radioimmunoassay, reverse phase high pressure liquid chromatography (rp HPLC) and immunohistochemistry, we have identified gastrin releasing peptide-immunoreactivity (GRP-IR) in the rat retina. The concentration of GRP-IR in retinal extracts was 7.4 +/- 0.6 ng/g wet wt. (mean +/- S.E.M. n = 15). There was no significant difference between the levels of immunoreactivity in 12-h light and 12-h dark adapted retinae. rp HPLC analysis of retinal extracts demonstrated that two main immunoreactive components were present which corresponded in retention time to GRP10 (neuromedin C) and GRP14 (GRP14-27). A small amount of material also co-eluted with GRP27. Using immunohistochemistry, the immunoreactivity has been localised in the inner retinal layers. Immunoreactive somata were present in the proximal inner nuclear layer and in the ganglion cell layer. Fibre staining was present in laminae 2 and 4 of the inner plexiform layer. Somatal staining was increased by pretreatment of retinae with vincristine while the laminar staining was markedly reduced. These results demonstrate the existence of GRP-like peptides in the rat retina which has not previously been reported.

Animals↗
PubMed1988

Physical disability and social liminality: a study in the rituals of adversity.

Sociological research on the disabled has for the past 25 years made extensive use of a social deviance model to characterize the status of the physically impaired. The present article, which is based on a three-year anthropological investigation of the social relations of paraplegics and quadriplegics in the New York metropolitan area, argues that there are shortcomings in the deviance model and offers, instead, a model taken from the anthropological study of ritual. The disabled are viewed as being in a 'liminal' state, as in the liminal phases of rites of passages. They are persons having an undefined status: they are neither ill nor well, neither socially alive and active nor socially expunged and removed. The status of the disabled in American society and the symbolism of disability in American culture are reexamined within this framework. This perspective is extended to other types of deep adversity, such as acute loss of income and status or catastrophic illness.

Activities of Daily Living↗